Nucleosomal arrays consist of core histone octamer-DNA complexes spaced at ~200 bp intervals along a DNA molecule. They are the fundamental building blocks of chromosomal superstructures, and the substrates for all functional processes in the eukaryotic nucleus; e.g., transciption, replication, repair. Nucleosomal arrays in solution exist in equilibrium between unfolded, moderately folded, extensively folded and oligomerized conformational states. Formation of each of these states is mediated by the core histone N-termini, acting through multiple molecular mechanisms. Incorporation of linker histones into nucleosomal arrays to form chromatin arrays shifts the equilibrium towards the extensively folded and oligomerized states, and in the process dramatically alters the structure of the linker DNA that connects adjacent nucleosomes in the array. The objectives of the proposed research are to delineate and characterize the mechanisms by which the core histone N-termini and linker histones exert their effects on chromatin folding and transcription by eukaryotic RNA polymerases. Specifically, using length- and compositionally-defined nucleosomal and chromatin arrays assembled from pure DNA and histone components, in combination with an innovative technical approach that integrates analytical ultracentrifugation, quanititative agarose gel electrophoresis, and electrophoresis, and electron cryo-microscopy, Dr. Hansen proposes to: (1) characterize how selective removal and targeted acetylation of the core histone N-termini influence nucleosomal array folding and oligomerization, and to determine whether the N-termini form homotypic or heterotypic complexes with each other, (2) characterize how different linker histone domains and sequence isotypes influence chromatin folding and linker DNA structure, and (3) perform correlative in vitro transcription studies to determine the extent to which chromatin folding represses transcription by RNA polymerases II and III. The proposed experiments are strongly hypothesis driven, and directly address several long-standing paradigms relating to chromatin structure and function. These studies will help provide the foundation necessary to achieve the long-term goal of assembling functionally important genetic loci in vitro entirely from pure histone and non-histone components.